The InlB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells

InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco-2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells. InlB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non-invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non-invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes , uptake of InlB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.     

Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors

Abstract: The large intracellular loop (IL) of the γ₂ subunit of the cloned human γ-aminobutyric acid_A (GABA_A) receptor (γ₂IL) was expressed in bacteria as glutathione- S -transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ₂IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABA_A/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000-M_r peptide band in immunoblots of affinity-purified GABA_A receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABA_A receptors in various brain regions.     

Rapid Phosphorylation of Phospholipase Cγ 1 by Brain-Derived Neurotrophic Factor and Neurotrophin-3 in Cultures of Embryonic Rat Cortical Neurons

Abstract: Phospholipase Cγ1 (PLC-γ1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron-like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC-γ1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on phosphorylation of PLC-γ1 in primary cultures of embryonic rat brain cells. BDNF and NT-3 stimulated the phosphorylation of PLC-γ1, followed by hydrolysis of PI. The stimulation of PLC-γ1 phosphorylation occurred within 20 s after addition of BDNF or NT-3 and lasted up to 30 min, with a peak after 4 min. ED₅₀ values were similar for BDNF and NT-3, with τ25 ng/ml. Phosphorylation of PLC-γ1 by BDNF and NT-3 was found in cultures from all major brain areas. K-252b, a compound known to inhibit selectively neurotrophin actions by interfering with the phosphorylation of trk -type neurotrophin receptors, prevented the BDNF- and NT-3-stimulated phosphorylation of PLC-γ1. Receptors of the trk type were coprecipitated with anti-PLC-γ1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT-3 seems to be neuron specific because no phosphorylation of PLC-γ1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT-3, and they indicate that the transduction mechanism of BDNF and NT-3 in the brain involves rapid phosphorylation of PLC-γ1 followed by PI hydrolysis.     

Cell invasion of Yersinia pseudotuberculosis by invasin and YadA requires protein kinase C, phospholipase C-γ1 and Akt kinase

The outer membrane proteins YadA and invasin of Yersinia pseudotuberculosis promote invasion into mammalian cells through β₁-integrins and trigger the production of interleukin (IL)-8. FAK, c-Src and the PI3 kinase were previously found to be important for both YadA- and invasin-promoted uptake. Here, we demonstrate that two different downstream effectors of PI3 kinase, Akt and phospholipase Cγ1 are required for efficient cell invasion. Inhibition of Akt or phospholipase C-γ (PLC-γ)1 by pharmaceutical agents as well as reduced expression of the isoforms Akt1 and Akt2, and of PLC-γ1 by RNA interference decreased entry of YadA- and Inv-expressing bacteria significantly. In addition, we report that the conventional protein kinases C (PKC)α and -β, positioned downstream of PLC-γ1, are activated upon Inv- or YadA-promoted cell entry. They colocalize with intracellular bacteria and their depletion by siRNA treatment also resulted in a strong reduction of cell entry. In contrast, neither Akt nor PLC-γ1, and the PKCs are essential for YadA- and Inv-mediated IL-8 synthesis and release. We conclude that YadA and invasin of Y. pseudotuberculosis both trigger similar signal transduction pathways during integrin-mediated phagocytosis into epithelial cells, which lead to the activation of Akt, PLC-γ1, PKCα and -β downstream of PI3 kinase, separate from the MAPK-dependent pathway that triggers IL-8 production.     

InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase

The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.     

Surface free energies of oral streptococci and their adhesion to solids

Abstract The adhesion of 3 strains of oral streptococci from a buffered suspension onto 3 different solid substrata was studied. Representative strains of streptococci were selected on the basis of their surface free energy ( γ _b), namely Streptococcus mitis L1 ( γ _b= 37 mJ·m⁻²), Streptococcus sanguis CH3 (95 mJ·m⁻²) and Streptococcus mutans NS (117 mJ·m⁻²). Solid substrata were also selected on basis of their surface free energy ( γ _s), and included polytetrafluorethylene ( γ _s= 20 mJ·m⁻²), polymethylmethacrylate (53 mJ·m⁻²) and glass (109 mJ·m⁻²). Bacterial adhesion was measured as the number of bacteria adhering per cm² at equilibrium. Equilibrium was usually obtained within 20 min. S. sanguis CH3, having an intermediate surface free energy did not show a clear preference for any of the 3 solids. S. mitis L1, however, the lowest surface free energy strain, adhered in highest numbers to the low energy solid PTFE, whereas the highest γ _b strain, S. mutans NS, adhered in highest numbers to the highest γ _s solid, glass. Calculation of the interfacial free energy of adhesion ( ΔF _adh) for each bacterial strain showed that this parameter was predictive of bacterial adhesion to solid substrata.     

The 213-amino-acid leucine-rich repeat region of the Listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Distinct protein patterns associated with Listeria monocytogenes InlA- or InlB-phagosomes

Internalization of Listeria monocytogenes into non-phagocytic cells is mediated by the interactions between the two bacterial invasion proteins InlA (internalin) and InlB and their cellular surface receptors E-cadherin and c-Met. To get an insight into all the cellular components necessary for uptake and early intracellular life, we undertook a global proteomic characterization of the early listerial phagosome in the human epithelial cell line LoVo. First, we proceeded to an immunocytochemical characterization of intracellular marker recruitment to phagosomes containing latex beads coated with InlA or InlB. E-cadherin and c-Met were, as expected, rapidly recruited to the phagosomal formation site. Phagosomes subsequently acquired the early endosomal antigen 1 (EEA1) and the lysosomal-associated membrane protein 1 (LAMP1), while presenting a more delayed enrichment of the lysosomal hydrolase cathepsin D. Early phagosomes devoid of lysosomal, endoplasmic reticulum and Golgi enzymatic activities could then be isolated by subcellular fractionation of LoVo cells. Two-dimensional gel electrophoresis (2DPAGE) revealed differences between the protein profiles of InlA- or InlB-phagosomes and those of early/late endosomes or lysosomes of naive LoVo cells. One major protein specifically recruited on the InlB-phagosomes was identified by mass spectrometry as MSF, a previously reported member of the septin family of GTPases. MSF forms filaments that co-localize with the actin cytoskeleton in resting cells and it is recruited to the entry site of InlB-coated beads. These results suggest that MSF is a putative effector of the InlB-mediated internalization of L. monocytogenes into host cells.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes

InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.     

Is the large plasmid of 120 MDa in Shigella sonnei composed of two DNA segments—90 and 30 MDa?

Abstract The reversibility of adhesion of 3 representative strains of oral streptococci from a phosphate-buffered suspension onto 5 different solid substrata was studied.
Streptococcus mitis T9 (surface free energy γ_b= 39 mJ · m⁻²). Streptococcus sanguis CH3 (γ_b= 95 mJ · m⁻²) and Streptococcus mutans NS (γ_b= 117 mJ · m⁻²) were selected on basis of their surface free energy. Solid substrata were employed with a surface free energy γ_s ranging from 20 mJ · m⁻² for polytetrafluorethylene to 109 mJ · m⁻² for glass. Bacterial suspensions containing 2.5 × 10⁹ cells per ml were incubated with 2 samples of each substratum. After 1 h the number of adhering bacteria was evaluated on one sample, while the second sample was kept for another hour at a 10-fold lower bacterial concentration. Bacteria with a low surface free energy desorbed only from substrata with a high surface free energy, while bacteria with a high surface free energy desorbed from substrata with a low surface free energy. Thus low energy bacterial strains adhered reversibly to high energy substrata and vice versa. Similar observations were made with polystyrene particles. Calculation of the interfacial free energy of adhesion (Δ F _adh) for each bacterial strain as well as for the polystyrene particles showed that a reversible adhesion was associated with a positive Δ F _adh, denoting unfavourable adhesion conditions upon a thermodynamic basis.     

MICROHETEROGENEITY OF BRAIN CYTOPLASMIC AND SYNAPTOPLASMIC ACTINS

Abstract— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two-dimensional gels and one-dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S₁ and S₂, with similar DNase I affinity were also isolated. S₁1 and S₂ have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S₁ and S₂ is 3-fOld greater in synaptosomes when compared to similar non-synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I-actin complex.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+-Dependent Proteolysis

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D₁ DA antagonist SCH 23390 (10⁻⁶ M ) but not by the D₂ antagonist sulpiride (10⁻⁵ M ). The D₁ agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D₂ agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D₁-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca²⁺-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca²⁺ channel blocker Co²⁺. The data suggest that DA stimulates a D₁-like DA receptor, which increases the influx of Ca²⁺ and activates the Ca²⁺-dependent proteolysis of γPKC.     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

FbpA, a novel multifunctional Listeria monocytogenes virulence factor

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.     

Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor-Induced Differentiation of PC12 Cells

Abstract: Phospholipase C γ1 (PLC-γ1) is phosphorylated on treatment of cells with nerve growth factor (NGF). To assess the role of PLC-γ1 in mediating the neuronal differentiation induced by NGF treatment, we established PC12 cells that overexpress whole PLC-γ1 (PLC-γ1PC12), the SH2-SH2-SH3 domain (PLC-γ1SH223PC12), SH2-SH2-deleted mutants (PLC-γ1ΔSH22PC12), and SH3-deleted mutants (PLC-γ1ΔSH3PC12). Overexpressed whole PLC-γ1 or the SH2-SH2-SH3 domain of PLC-γ1 stimulated cell growth and inhibited NGF-induced neurite outgrowth of PC12 cells. However, cells expressing PLC-γ1 lacking the SH2-SH2 domain or the SH3 domain had no effect on NGF-induced neuronal differentiation. Overexpression of intact PLC-γ1 resulted in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2-SH2-SH3 domain of PLC-γ1 did not alter total inositol phosphate accumulation. To investigate whether the SH2-SH2-SH3 domain of PLC-γ1 can mediate the NGF-induced signal, tyrosine phosphorylation of the SH2-SH2-SH3 domain of PLC-γ1 on NGF treatment was examined. The SH2-SH2-SH3 domain of PLC-γ1 as well as intact PLC-γ1 could be tyrosine-phosphorylated on NGF treatment. These results indicate that the overexpressed SH2-SH2-SH3 domain of PLC-γ1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC-γ1 but to the SH2-SH2-SH3 domain of PLC-γ1.     

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A-R) is alternatively spliced. The long variant (γ_2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca²⁺/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ_2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K _m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ_2L subunit has been shown to be necessary for ethanol potentiation of the GABA_A-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.